A method for in vitro culture of taro sprouts in a substrate

Abstract

This invention relates to the field of crop cultivation technology, specifically to a method for in vitro culture of taro sprouts in a substrate. The method involves selecting uniform, disease-free taro tubers weighing 15-25g each as seed tubers and pre-treating them with sterilization. After sprouting, the seed tubers are cut. When the new sprouts are 1-2cm long and have 2-3 roots, the main bud is cut off along the lower edge of the new roots. This main bud is placed in a vegetable seedling substrate for seedbed cultivation to obtain seedlings, with temperature and humidity controlled during the cultivation process. When two new leaves have grown and the plant height is 8-10cm, the seedlings are transplanted for field planting. After transplanting, soil moisture is controlled to complete the planting process. This invention, by cutting off and culturing the initially sprouted taro sprouts, can restore and cultivate them into complete plants, achieving disease removal and rejuvenation. This invention can cut off pathogens carried by the taro tubers at the source, effectively reducing the spread of seed-borne diseases in seed tubers, increasing the health rate of offspring seed tubers, and simultaneously ensuring that the proliferation coefficient of daughter tubers meets the needs of large-scale propagation of healthy seed tubers.

Description

Technical Field

[0001] This invention relates to the field of crop cultivation technology, specifically to a method for in vitro culture of taro sprouts in a substrate. Background Technology

[0002] Taro (Colocasia esculenta L. Schott) is a perennial monocotyledonous plant belonging to the genus Colocasia in the family Araceae. It has underground corms and is often cultivated as an annual. China ranks second in the world in total taro production. The main edible part of taro is the underground corm, which is rich in starch, accounting for 60-70% of the total dry matter.

[0003] In production, taro is mainly propagated using corms. Long-term use of self-saved seeds leads to the accumulation of diseases and severe degeneration of the taro plants. Taro dry rot, also known as taro wilt, is a seed-borne disease caused by Fusarium solani. The pathogen mainly parasitizes the vascular bundles of the taro stem. In the early stages of the disease, growth slows down, and older leaves turn yellow rapidly. Severely affected plants show poor growth, with stems and leaves turning yellowish-green, and premature drying or lodging in autumn. Peeling back the corm reveals a reddish-brown flesh layer with small red spots visible upon cross-section. In severe cases, large pieces of flesh turn reddish-brown, causing dry rot or hollowing. Current technology often uses virus-free tissue culture to purify and rejuvenate taro germplasm. However, virus-free tissue culture seedlings have low transplant survival rates, poor field competitiveness, and produce small, fewer corms in the same year. Furthermore, the tissue culture process may involve other variations and carries the risk of virus escape, failing to meet the needs of large-scale taro germplasm purification and rejuvenation in production.

[0004] The lack of technology for propagating healthy taro seedlings is a technological bottleneck restricting the sustainable development of the taro industry. Therefore, there is an urgent need to develop a method suitable for the large-scale propagation and production of healthy taro seedlings to ensure seed safety for the taro industry. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for in vitro culture of taro sprouts in a substrate. This method ensures the healthy growth of taro sprout regenerated seedlings throughout their entire growth cycle, while simultaneously providing a maximum number of healthy seed taro plants that meet seed production requirements in the same year. This method is characterized by its effective disease control, ease of operation, low cost, and scalability.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] A method for in vitro culture of taro sprouts using a substrate, which utilizes newly sprouted taro shoots to regenerate and culture them in vitro into complete, disease-free taro seedlings, specifically includes the following steps:

[0008] S1. Seed tuber selection and treatment: Initial field selection is carried out on the seed field to remove plants infected with mosaic virus; before cultivation, select tuber tubers that are uniform in appearance, free of disease spots, and weigh 15-25g each as seed tubers; soak the seed tubers in Amistar bactericide solution, then remove them, dry the surface moisture, and set aside for use.

[0009] S2. In vitro culture of taro sprouts in a substrate: After the seed taro is sprouted, the sprouts are cut off and the cut-off sprouts are cultured in a seedbed in a vegetable seedling substrate to obtain disease-free taro seedlings;

[0010] S2-1. Sprouting and cutting the bud: Sprouting is carried out on the seed tubers that have been soaked. During sprouting, the seed tubers are completely covered with vegetable seedling substrate, and the thickness of the vegetable seedling substrate covering the seed tubers is 2-3cm. The sprouting time is controlled within 1 month. When the new buds of the seed tubers are 1-2cm long (buds longer than 3cm are no longer used) and have 2-3 roots, the main bud is cut off along the lower edge of the new roots of the seed tubers, keeping the new roots and not damaging the new roots when cutting the buds, for subsequent seedling cultivation.

[0011] S2-2, Seedling cultivation: Select a perforated tray, arrange the roots of the cut main bud in a tray with the bud tip facing upwards, and bury it in the perforated tray containing vegetable seedling substrate. The vegetable seedling substrate should completely cover the roots but not the bud tip to obtain substrate-cultured taro sprouts.

[0012] Arrange the tissue-cultured taro sprouts in the seedbed neatly and place them in an environment with a temperature of 18-25℃ for seedling cultivation. During the seedling cultivation period, the moisture content of the vegetable seedling substrate should be maintained at 60wt%-70wt%. When the tissue-cultured taro sprouts have grown two flat new leaves and the plant height is 8-10cm, disease-free taro seedlings are obtained and then transplanted into the field and planted in the field.

[0013] S3. Transplant the disease-free taro seedlings to the field. Immediately after transplanting, water them and cover them with shade netting. Cover them with shade netting all day for 7-10 days after transplanting to maintain the soil moisture content of the seedbed at 50wt%-60wt%. Water them as needed in the morning and evening.

[0014] Vegetable seedling substrate is used for germination, cultivation and transplanting. Germination has low requirements for substrate, just loose and moist. The effective nutrients during the cultivation stage can supply the seedlings for about one month. In the later stage, loose substrate is used for transplanting to ensure that the roots are not damaged and to facilitate root development after transplanting.

[0015] Furthermore, in S1, the seed tubers were soaked in a 2000-fold diluted solution of Amistar for 5-15 minutes. Amistar's main components are a suspension composed of azoxystrobin and difenoconazole, possessing both protective and curative activities, and are highly suitable for resistance management and integrated disease control. It has a broad fungicidal spectrum, showing good efficacy against diseases caused by ascomycetes, basidiomycetes, deuteromycetes, and oomycetes, such as powdery mildew, downy mildew, rust, blight, rice blast, smut, leaf spot, and root and stem diseases.

[0016] Furthermore, prepare germination seedbeds in a 6-meter-high greenhouse, each seedbed being 1.6-1.8 meters wide. Level the seedbeds beforehand and water them thoroughly. Spread the soaked seed tubers evenly on the seedbeds, stacking them no more than 3 layers high. Cover the seed tubers with vegetable seedling substrate, with a thickness of 2-3 cm. Spray the substrate thoroughly with water, and then cover the surface of the vegetable seedling substrate with a transparent plastic film to retain moisture and heat. Every 7-10 days, uncover the film to check the germination status of the seed tubers and the moisture content of the vegetable seedling substrate, and promptly sort and remove any rotten seed tubers.

[0017] Furthermore, in S2-1, bud cutting is performed using knives and mats sterilized with 75% alcohol;

[0018] In S2-2, 72-well or 50-well perforated trays are used for seedling cultivation, and the seedling cultivation time is 2-3 weeks.

[0019] Furthermore, in S2-2, during the seedling cultivation period, bamboo frames are erected on the seedling beds in the greenhouse. If the temperature inside the greenhouse is below 18℃, small arched sheds for seedling cultivation need to be erected. If the temperature inside the greenhouse exceeds 25℃ during the day, a shading net with a 70% shading rate needs to be covered on the small arched sheds, and the plastic film at both ends of the small arched sheds needs to be opened to maintain ventilation. If the temperature inside the greenhouse is below 15℃ at night, an insulation blanket needs to be laid outside the small arched sheds for insulation.

[0020] Furthermore, before transplanting S3 seedlings to the field, the field needs to be treated with calcium cyanamide, compound fertilizer should be applied in the field, and ridges should be formed after plowing; the ridge width should be 0.7-0.9m, the ridge height should be 20-30cm, and the furrow width should be 0.2-0.5m; the ridge surface should be covered with black fully biodegradable mulch film with a thickness of 0.01mm, a width of 1.2m, and a weather resistance period of 80-150 days.

[0021] Furthermore, the field planting described in S3 is double-row planting, with holes punched on the ridge at a plant spacing of 30-40cm and a hole depth of 10-15cm; the holes are filled with conventional vegetable seedling substrate, and after watering thoroughly, the substrate is replenished again to be level with the ridge surface.

[0022] Beneficial Technical Effects: This invention selects taro tubers with uniform appearance, no disease spots, and a weight within a specific range of 15-25g as seed tubers. After soaking and sterilizing, the seed tubers are fully covered with a seedling substrate and sprouted under certain conditions for a period of time. Then, the main buds with new shoots 1-2cm in length and 2-3 roots are cut off and placed in a greenhouse for seedling cultivation using a seedling substrate. Strict control is maintained over the cultivation time and water management during seedling cultivation. After transplanting, soil moisture is controlled for 1-2 weeks after transplanting to ensure successful plant development. The taro sprout substrate in vitro culture method of the present invention can restore and cultivate complete plants by cutting off the initially germinated taro sprouts, achieving the effect of disease removal and rejuvenation; the growth performance after transplanting is good, and the incidence of stem rot in the field is low; the healthy seed tubers (grade 0-1) rate of the planted tubers reaches more than 83%; the present invention can cut off the pathogens carried by the taro corm from the source, which can effectively reduce the spread of seed-borne diseases in seed tubers, improve the health rate of offspring seed tubers, and at the same time, the tuber proliferation coefficient can meet the needs of large-scale propagation of healthy seed tubers. Attached Figure Description

[0023] Figure 1 Images of S2-1 seed tubers after sprouting;

[0024] Figure 2 Image of the main bud obtained by cutting the buds of seed tubers after S2-1 sprouting;

[0025] Figure 3 Images of taro sprouts cultured in substrate during the S2-2 seedling cultivation process;

[0026] Figure 4 Image of substrate tissue culture seedlings (i.e. disease-free taro seedlings) obtained from S2-2 seedling cultivation;

[0027] Figure 5 Images show the field preparation before transplanting S3 seedlings and the field planting after transplanting. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. Techniques and methods known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques and methods should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0030] Experimental methods not specified in the following examples are generally performed according to national standards; if there is no corresponding national standard, they are performed according to general standard requirements or general methods.

[0031] The vegetable seedling substrate used below is a vegetable cultivation substrate, provided by Jiangsu Xingnong Substrate Technology Co., Ltd., product number 161102G0096N. For specific components, please refer to the Chinese invention patent with authorization announcement number CN103708952B.

[0032] Example 1

[0033] Selection of the size of taro bulbs (hereinafter referred to as seed bulbs)

[0034] From January 15 to March 9, 2022, taro sprout substrate in vitro culture was conducted in a smart greenhouse of Jiangsu Academy of Agricultural Sciences to carry out sprouting experiments on taro tissue culture seedlings. The taro variety was Jingjiang Xiangsha taro.

[0035] The specific implementation steps are as follows:

[0036] S1. Selection and treatment of seed potatoes:

[0037] The seed production field was initially selected, and plants infected with mosaic virus were removed. Before cultivation, taro tubers with uniform appearance and no disease spots were selected and divided into four groups according to weight: <8g, 8-15g, 15-25g, and 25-40g, with 50 tubers in each group. They were packed in 40×60cm mesh bags and soaked in a 2000-fold diluted solution of Ammonium chloride for 10 minutes. After soaking, they were removed and dried for later use.

[0038] S2, Taro sprout substrate in vitro sprouting:

[0039] A layer of conventional vegetable seedling substrate about 3cm thick was laid on the soil of the intelligent greenhouse. The vegetable seedling substrate was moistened with water. The pre-soaked Jingjiang Xiangsha taro bulbs were spread flat on the substrate, and then another layer of vegetable seedling substrate about 3cm thick was covered on top. A layer of plastic film was then placed on the outside, and finally a heat preservation blanket was placed on top for germination. After one month of germination, the bulbs were taken out every 7 days to check the changes in germination and to detect any rotten bulbs. The results are shown in Table 1.

[0040] Table 1. Dynamic changes in bulb size on the sprouting of Jingjiang sweet potato.

[0041]

[0042] As shown in Table 1, by March 9th, taro bulbs weighing less than 8g exhibited slow germination, weak root development, and a 30.0% rate of empty bulb rot. Bulbs weighing 25-40g showed a 12.0% rate of soft, rotten tails. Bulbs weighing 8-15g had an 86.0% bud germination rate, with 76.0% of the buds approximately 1cm in length and developing 1-3 roots. Bulbs weighing 15-25g had a 92.0% bud germination rate, an 82.0% root development rate, a 4.0% bulb rot rate, and relatively uniform bud lengths of 1-2cm. Considering all factors, bulbs weighing 15-25g are suitable for in vitro taro cultivation using taro sprout substrate.

[0043] Example 2

[0044] In 2022, substrate-based seedling cultivation of Jintan Red Taro, Jingjiang Xiangsha Taro, and Haimen Xiangsha Taro was conducted at the Liuhe Base of the Jiangsu Academy of Agricultural Sciences. The in vitro culture method for taro sprouts of different varieties, utilizing newly sprouted taro shoots, was used for in vitro regeneration and culture to produce complete, disease-free taro seedlings. The specific steps included:

[0045] S1. Selection and treatment of seed potatoes:

[0046] The seed field is initially selected to remove plants infected with mosaic virus. Before cultivation, 500 seed tubers of each variety are selected with a uniform appearance, no disease spots, and a single weight of 15-25g. The seed tubers are soaked in a 2000-fold diluted solution of Aminopyrine for 10 minutes, then removed and dried for later use.

[0047] S2, Taro sprout substrate in vitro culture:

[0048] After the seed tubers are sprouted, the sprouts are cut off, and the cut-off sprouts are cultured in a seedbed in a conventional vegetable seedling substrate to obtain seedlings;

[0049] S2-1. Sprouting and Cutting: Prepare sprouting beds in a 6-meter-high greenhouse, each bed 1.6-1.8m wide; level the beds beforehand and water them thoroughly; evenly spread the soaked seed tubers on the beds, stacking them no more than 3 layers high; cover the seed tubers with vegetable seedling substrate, about 3cm thick, and spray with water; then cover the surface of the vegetable seedling substrate with a transparent plastic film to retain moisture and heat; every 7-10 days, uncover the film to check the sprouting of the seed tubers and the moisture content of the vegetable seedling substrate, and promptly sort and remove any rotten seed tubers;

[0050] When the new shoots on the seed tuber grow to 1-2cm in length and have developed 2-3 roots (see details...), Figure 1Disinfect the knife and cutting board with 75% alcohol before cutting the bud. When cutting the bud, cut the main bud along the lower edge of the newly sprouted root of the seed tuber (see details). Figure 2 Be careful not to damage the roots;

[0051] S2-2, Seedling Cultivation: Select 72-cell or 50-cell seedling trays. Arrange the cut main bud roots neatly in the trays with the bud tip facing upwards, then place them into the trays filled with vegetable seedling substrate. The bud tip should be exposed, and the roots should be completely buried in the substrate. This yields substrate-cultured taro sprouts (see details below). Figure 3 );

[0052] Arrange the tissue-cultured taro sprouts in the seedbed neatly and place them in a greenhouse at a temperature of 18-25℃ for 2-3 weeks of seedling cultivation. During the seedling cultivation period, keep the vegetable seedling substrate moist (60wt%-70wt%). During the seedling cultivation period: set up bamboo frames for the seedbed in the greenhouse. If the temperature inside the greenhouse is below 18℃, a small arched greenhouse is required. If the temperature inside the greenhouse exceeds 25℃ during the day, cover the small arched greenhouse with a 70% shading net and open the plastic film at both ends of the small arched greenhouse to maintain ventilation. If the temperature inside the greenhouse is below 15℃ at night, lay an insulation blanket outside the small arched greenhouse for insulation. During the seedling cultivation period, spray water evenly every morning and evening to keep the substrate moist.

[0053] When the taro sprouts from the substrate culture have grown two new, flat leaves and are 8-10 cm tall, disease-free taro seedlings are obtained (hereinafter referred to as substrate culture seedlings; see details below). Figure 4 ), and then proceed with subsequent field transplanting and field planting;

[0054] S3. Transplant the tissue culture seedlings to the field. Before transplanting, the field needs to be treated with calcium cyanamide. Apply 50 kg / mu of N-15-P-15-K-15 compound fertilizer to the field, plow, and then create ridges. The ridges should be about 0.8 m wide, 25 cm high, and the furrows about 0.3 m wide. Cover the ridges with black fully biodegradable mulch (see details). Figure 5 (Left image) The plastic film is 0.01mm thick, 1.2m wide, and has a weather resistance period of about 100 days;

[0055] For double-row planting, make holes in the ridges at a spacing of 30-40cm between plants, with a hole depth of 10-15cm. Fill the holes with vegetable seedling substrate, water thoroughly, and then fill the holes again with substrate until level with the ridge surface. After transplanting, proceed with field planting as follows: Figure 5 As shown in the right picture, water the seedlings immediately after transplanting, set up a trellis and cover them with a shade net. Cover them with a shade net all day for 7-10 days after transplanting, and water them as needed in the morning and evening according to the soil moisture in the seedbed.

[0056] The specific results are shown in the following data.

[0057] Table 2. Survival rate of substrate tissue culture seedlings of Jintan Red Taro, Jingjiang Fragrant Taro, and Haimen Fragrant Taro in 2022

[0058] variety Taro sprout germination rate (%) Transplant survival rate (%) Jintan Red Taro 90.28 92.15 Haimen fragrant taro 93.06 95.22 Jingjiang Fragrant Taro 77.78 93.53

[0059] Table 3. Field growth dynamics and disease incidence of tissue culture seedlings in three substrates in 2022.

[0060]

[0061]

[0062] Table 4 Theoretical yield of tissue culture seedlings from three different substrates in 2022

[0063]

[0064] Table 5. Health grading of taro seedlings from substrate tissue culture of three varieties in 2022

[0065]

[0066] (Note: Grading standards for diseased taro: Grade 0: When the taro is cut crosswise, the flesh is white and free of impurities; Grade 1: When the taro is cut crosswise, the flesh is slightly yellow and free of impurities; Grade 2: When the taro is cut crosswise, the flesh is slightly yellow and occasionally has reddish-brown spots near the skin; Grade 3: When the taro is cut crosswise, the flesh is yellowish and has obvious reddish-brown filaments.)

[0067] The data above show that the survival rate of the three multi-seed taro varieties, namely Jintan Red Taro, Jingjiang Xiangsha Taro, and Haimen Xiangsha Taro, after transplanting seedlings obtained by in vitro culture of taro sprouts using the substrate of this invention all exceeded 92.15% (Table 2). The incidence of stem rot in the field was 6.0-20.0% (Table 3). The number of effective seed taro harvested in the same year exceeded 1:10 (Table 4), and the rate of healthy seed taro (grade 0-1) was 83.64-88.46% (Table 5).

[0068] Comparative Experiment 1

[0069] In 2021, a comparative experiment was conducted at the Liuhe Base of Jiangsu Academy of Agricultural Sciences on taro sprout substrate in vitro culture (hereinafter referred to as substrate tissue culture seedling) and bulb sowing. The taro variety was Jintan Red Taro, and the seed tubers of Jintan Red Taro were selected with a size of 15-25g.

[0070] The substrate tissue culture method is the same as in Example 2.

[0071] The seed bulbs are planted directly in the field using seed tubers (which have undergone sterilization treatment in S1). The field planting method is the same as S3 in Example 2.

[0072] Table 6. Theoretical yield of Jintan red taro tissue culture seedlings and bulbs.

[0073]

[0074] Table 7 Health Grading of Different Types of Tissue Culture Seedlings of Jintan Red Taro

[0075]

[0076] (Note: Grading standards for diseased taro: Grade 0: When the taro is cut crosswise, the flesh is white and free of impurities; Grade 1: When the taro is cut crosswise, the flesh is slightly yellow and free of impurities; Grade 2: When the taro is cut crosswise, the flesh is slightly yellow and occasionally has reddish-brown spots near the skin; Grade 3: When the taro is cut crosswise, the flesh is yellowish and has obvious reddish-brown filaments.)

[0077] As shown in Tables 6 and 7, in the same experimental plot, using the same variety of Jintan Red Taro, compared to bulb sowing, the Jintan Red Taro substrate tissue culture seedlings of this invention retained the edge rhizome. During the harvest period, yield measurements showed that each plant yielded 15.4 effective seed tubers (single bulb weight ≥30g) suitable for seed production in the second year, significantly more than the 11.0 from bulb sowing. The theoretical yield reached 1343.17 kg / mu, slightly higher than bulb sowing by 3.4%. The healthy seed tuber (grade 0-1) rate of the substrate tissue culture seedlings reached 90.91%, while the rate of healthy seed tubers (grade 0-1) from bulb sowing was 61.82% (Table 7). The in vitro taro sprout substrate culture method of this invention can effectively reduce the spread of seed-borne diseases in seed tubers, improve the health rate of offspring seed tubers, and the seed tuber proliferation coefficient can meet the needs of healthy seed tuber propagation.

[0078] Comparative Experiment 2

[0079] In 2022, a comparative experiment was conducted at the Liuhe Base of Jiangsu Academy of Agricultural Sciences on taro sprouts under different conditions for in vitro culture in substrate (hereinafter referred to as substrate tissue culture seedlings). The taro variety was Jintan Red Taro, and the seed bulb weight was 15-25g.

[0080] The specific implementation steps are as follows:

[0081] (1) Select 500 Jintan red taro seed bulbs with neat appearance and no obvious disease spots, soak them in a solution of AmiMiaoShou diluted 2000 times for 10 minutes, take them out and dry them for later use.

[0082] (2) Prepare germination beds in the greenhouse, each bed 1.6-1.8m wide; level the beds beforehand and water them thoroughly; spread the soaked seed tubers evenly on the beds, stacking them no more than 3 layers high; cover the tubers with conventional vegetable seedling substrate, about 3cm thick, and spray with water; cover the substrate surface with a transparent plastic film to retain moisture and warmth; every 7-10 days, uncover the film to check the germination of the tubers and the moisture content of the substrate, and promptly sort and remove any rotten tubers; (the above two steps are the same as S1 and S2-1 in Example 2)

[0083] (3) In late April, the sprouted seed bulbs were sorted and divided into two groups according to the sprout length: ① Sprouts with a length of 1-2 cm and 2-3 new roots were cultured in a substrate and treated as S2-2 and S3 in Example 2 (hereinafter referred to as substrate culture seedlings); ② Sprouts with a length of 3-5 cm and ≥3 new roots were directly transplanted to the field and treated as S3 in Example 2 (hereinafter referred to as direct seeding of taro sprouts).

[0084] Table 8. Survival rate of different types of tissue culture seedlings of Jintan Red Taro after transplanting.

[0085] Tissue culture seedling types Germination rate (%) Transplant survival rate (%) Seedling survival rate (%) ① Substrate tissue culture seedlings 90.28 92.80 90.20 ② Bud cutting live broadcast - 90.30 81.18

[0086] Table 9. Field growth dynamics and disease incidence of different types of tissue culture seedlings of Jintan Red Taro.

[0087]

[0088] Table 10 Theoretical Yield of Different Types of Tissue Culture Seedlings of Jintan Red Taro

[0089]

[0090] Table 11 Health Grading of Different Types of Tissue Culture Seedlings of Jintan Red Taro

[0091]

[0092] (Note: Grading standards for diseased taro: Grade 0: When the taro is cut crosswise, the flesh is white and free of impurities; Grade 1: When the taro is cut crosswise, the flesh is slightly yellow and free of impurities; Grade 2: When the taro is cut crosswise, the flesh is slightly yellow and occasionally has reddish-brown spots near the skin; Grade 3: When the taro is cut crosswise, the flesh is yellowish and has obvious reddish-brown filaments.)

[0093] The data above shows that the longer the taro sprouts are, the more roots they will have. However, the amount of pathogens accumulated in the taro sprouts will also increase, and the disease rate will rise. For seed taro that has been sterilized with S1, the smaller the size of the main bud that meets the conditions for bud regeneration and is controlled for cultivation, the shorter the early cultivation time will be, and the better the quality of the taro will be. Therefore, it is not recommended to use main buds larger than 3cm for regeneration. After the seed tubers of Jintan Red Taro were sprouted, they were classified according to the sprout length. Compared with the direct sowing treatment of cut sprouts, the present invention uses the in vitro culture method of taro sprout substrate for substrate tissue culture seedlings. The survival rate of the substrate tissue culture seedlings after transplanting all exceeded 90.3% (Table 8). The two types of tissue culture seedlings sprouted in the same batch showed high growth synchronicity. The growth performance of the substrate tissue culture seedlings after transplanting was better than that of the direct-sown seedlings, and the incidence of stem rot in the field was slightly lower than that of the direct-sown seedlings (Table 9). The number of effective seed tubers harvested in the same year for both treatments exceeded 1:10 (Table 10), but the healthy seed tuber (0-1 grade) rate of the substrate tissue culture seedlings reached 88.46%, which was better than that of the direct-sown seedlings (Table 11).

[0094] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for in vitro culture of taro sprouts in a substrate, characterized in that, The process of using newly sprouted taro shoots to regenerate and culture complete, disease-free taro seedlings in vitro includes the following steps: S1. Seed tuber selection and treatment: Initial field selection is carried out on the seed field to remove plants infected with mosaic virus; before cultivation, select tuber tubers that are uniform in appearance, free of disease spots, and weigh 15-25g each as seed tubers; soak the seed tubers in Amistar bactericide solution, then remove them, dry the surface moisture, and set aside for use. S2. In vitro culture of taro sprouts in a substrate: After the seed taro is sprouted, the sprouts are cut off and the cut-off sprouts are cultured in a seedbed in a vegetable seedling substrate to obtain disease-free taro seedlings; S2-1. Sprouting and cutting the bud: Sprouting the seed tubers that have been soaked is carried out. During sprouting, the seed tubers are completely covered with vegetable seedling substrate, and the thickness of the vegetable seedling substrate covering the seed tubers is 2-3cm. The sprouting time is controlled within 1 month. When the new buds of the seed tubers are 1-2cm long and have 2-3 roots, the main bud is cut off along the lower edge of the new roots of the seed tubers, retaining the new roots and not damaging the new roots when cutting the buds, for subsequent seedling cultivation. S2-2, Seedling cultivation: Select a perforated tray, arrange the roots of the cut main bud in a tray with the bud tip facing upwards, and bury it in the perforated tray containing vegetable seedling substrate. The vegetable seedling substrate should completely cover the roots but not the bud tip to obtain substrate-cultured taro sprouts. The sown taro sprouts in the substrate are neatly arranged in a seedling bed and placed in an environment with a temperature of 18-25℃ for seedling cultivation. During the seedling cultivation period, the moisture content of the vegetable seedling substrate should be maintained at 60wt%-70wt%. When the taro sprouts in the substrate have grown two flat new leaves and the plant height is 8-10cm, disease-free taro seedlings are obtained and then transplanted into the field and planted in the field. S3. Transplant the disease-free taro seedlings to the field. Immediately after transplanting, water them and cover them with shade netting. Cover them with shade netting all day for 7-10 days after transplanting to maintain the soil moisture content of the seedbed at 50wt%-60wt%. Water them as needed in the morning and evening.

2. The method for in vitro culture of taro sprouts according to claim 1, characterized in that, In S1, the seed tubers are soaked in a 2000-fold diluted solution of Aminopyrine for 5-15 minutes.

3. The method for in vitro culture of taro sprouts according to claim 1, characterized in that, The germination method described in S2-1 is as follows: Germination beds are prepared in a 6-meter-high greenhouse, each bed being 1.6-1.8 meters wide. The beds are leveled and thoroughly watered beforehand. The soaked seed tubers are evenly spread on the seed beds, stacked no more than 3 layers high. The seed tubers are covered with a vegetable seedling substrate, with a thickness of 2-3 cm. The substrate is then thoroughly watered. A transparent plastic film is then placed over the surface of the vegetable seedling substrate to retain moisture and warmth. Every 7-10 days, the film is removed to check the germination status of the seed tubers and the moisture content of the vegetable seedling substrate, and any rotten seed tubers are promptly sorted and removed.

4. The method for in vitro culture of taro sprouts according to claim 1, characterized in that, In S2-1, bud cutting is performed using knives and mats sterilized with 75% alcohol; in S2-2, seedling cultivation is carried out using perforated trays with 72 or 50 holes.

5. The method for in vitro culture of taro sprouts according to claim 1, characterized in that, During the seedling cultivation period described in S2-2, bamboo frames are erected on the seedling beds inside the greenhouse. If the temperature inside the greenhouse is below 18℃, small arched sheds for seedling cultivation are required. If the temperature inside the greenhouse exceeds 25℃ during the day, a shading net with a 70% shading rate is required to cover the small arched sheds, and the plastic film at both ends of the small arched sheds should be opened to maintain ventilation. If the temperature inside the greenhouse is below 15℃ at night, an insulation blanket is required to be laid outside the small arched sheds for insulation.

6. The method for in vitro culture of taro sprouts according to claim 1, characterized in that, Before transplanting to the field, the field needs to be treated with calcium cyanamide, compound fertilizer should be applied in the field, and ridges should be made after plowing. The ridges should be 0.7-0.9m wide, 20-30cm high, and the furrows 0.2-0.5m wide. The ridges should be covered with black fully biodegradable mulch film with a thickness of 0.01mm, a width of 1.2m, and a weather resistance period of 80-150 days.

7. The method for in vitro culture of taro sprouts according to claim 1, characterized in that, For double-row planting, make holes on the ridge with a plant spacing of 30-40cm and a hole depth of 10-15cm; fill the holes with vegetable seedling substrate, water thoroughly, and then fill the holes again with substrate until they are level with the ridge surface.

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